Primary T-lymphocyte (T-cell) immunodeficiency is mainly attributable to defects in thymus development because the thymus provides a unique microenvironment for development and maintenance of broadly reactive and self-restricted T cells and a diverse peripheral T-cell repertoire. Primary T-cell immunodeficiency caused by thymic organogenetic and thymic stromal cell (TSC) developmental failure, such as DiGeorge syndrome (DGS) which is likely caused by a mutation in the Tbx1 gene and human nude (HN) which is caused by a mutation in the Foxn1 gene, can be rescued by regenerating a de novo ectopic thymus using an ex vivo three-dimensional TSC network. However, the source of a safe, stable, and effective donor thymus becomes a problem because thymectomy during cardiothoracic surgery will cause profound problems in the donor's immune system. In order to obtain a safe, stable, and effective TSC network, we propose generating a de novo ectopic thymus under the kidney capsule by using various stromal cells in a mouse model. We will test engineered Notch ligand and Foxn1 over-expressing skin fibroblasts and keratinocytes from normal or Tbx1 mutant mice for generating a functional de novo thymus for DGS therapy detailed in our Aim 1. We will identify specific phenotypic markers from putative thymic epithelial progenitors through our novel Foxn1Neo/Neo and Foxn1Neo/+ mice by G418-selection culture;and then isolate and expand the putative thymic epithelial progenitors from wild-type thymus based on the specific thymic epithelial progenitor phenotypic markers for testing the strategy of HN therapy detailed in our Aim 2. The comprehensive knowledge obtained from the proposed studies will lay the groundwork to extend the study to human subjects in the future, and lead to the development of practical strategies against T-cell immunodeficiency caused by primary thymic organogenetic and/or TSC developmental failure. PUBLIC HEALTH RELEVANCE: We will test various stromal cells, including Notch ligand and Foxn1 over-expressing skin fibroblasts and keratinocytes from wild-type or Tbx1 mutant mice and isolate and expand the putative thymic epithelial progenitors from wild-type mice, for generating a functional de novo thymus for DiGeorge syndrome and human nude therapies in a mouse model.